This investigation of the properties of cytoplasmic membrane proteins from B. megaterium KM focusses on two related but distinct areas. The first is a continuation of an investigation of the properties of membrane bound ATPase in both its bound and solubilized forms, including preparation of antibody to the purified ATPase. Physical and chemical properties of the solubilized and membrane bound enzymes will be studied including molecular weight determination using the ultracentrifuge and amino acid analysis. The conditions for rebinding of the purified, soluble ATPase to the cytoplasmic membrane will be determined. In particular we wish to find out whether an additional protein is required for rebinding, since it seems to be necessary in other membrane bound ATPase systems. If it is, we hope to purify and characterize it. The second part of the project involves studies on the spatial orientation of membrane proteins using both chemical and immunological methods. I125 and lactoperoxidase will be used to iodinate proteins in whole bacteria, protoplasts and isolated cytoplasmic membranes. Proteins iodinated under the three different conditions will be compared by separation on SDS polyacrylamide gel electrophoresis and we will pay particular attention to the conditions under which ATPase and flagellin are labelled to see whether these proteins are located on the outside or inside of the membrane. Antibodies to purified ATPase, flagella and flagellin, and whole cytoplasmic membranes will be prepared in rabbits. By comparison of the purified antibodies with antibodies to whole membranes we will determine whether ATPase or flagella/flagellin are major antigenic determinants of the cytoplasmic membrane. By testing to see which antibodies are absorbed to protoplasts, and which show lines of identity with whole membrane antisera we hope to obtain information on the spatial location of the major membrane antigens.